Has anyone tried using T7 or e.coli ligase instead of T4 ligase for ligating adapters? My lab uses custom adapters for amplicon sequencing on the MiSEQ. We have been having issues with adapter dimers and a strange "ladder" effect underneath our target band after barcoding PCR and after some investigation we think that it might be related to active T4 ligase in the PCR reaction. We use the 3' A-overhang of the PCR fragments to ligate on our adapters. Can you use T7 or other ligase types for TA ligations?? thanks for the help everyone!
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Ligation reaction should be cleaned up before PCR to reduce excess adapters, ligase and ligase buffer reagents carry over. Ligase is less likely cause of your observation because it would be deactivated very early in PCR temperatures and also it is active at low temperatures. T7 ligase also can be used as long as there are cohesive ends for ligation, but T4 is more robust than T7.
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The immune system’s power comes from its genetic diversity, allowing myriad threats to be neutralized through first recognizing foreign antigens. That diversity is also what makes the immune system so difficult to study. Recent advances in sequencing technology and computational biology, however, are giving researchers new tools to understand immune responses and immune-related diseases in greater detail.
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